This newly formed Project 3 utilizes state-of-the-art tools to analyze regulation of gene expression, including alterations in chromatin structure, in the brain's reward regions by opiates and cocaine. One objective is to characterize changes in mRNA levels in the NAc (nucleus accumbens) and ventral tegmental area (VTA) after chronic administration of opiates or cocaine and after varying periods of withdrawal. This endeavor will also include comparison of the effects of self-administered vs forced drug exposure. These analyses, which are ongoing, are already providing unique insight into the shifting patterns of gene expression changes that occur in the VTA-NAc during a course of drug treatment and recovery during withdrawal. A major advance over the past 4 years of the PPG has been the development of experimental protocols to study changes in chromatin structure within brain reward regions in rodent models. This includes chromatin immunoprecipitation (ChIP) assays as well as ChIP combined with DNA promoter microarrays, socalled "ChIP on chip" assays. Such studies of chromatin remodeling offer two major advances over conventional DNA expression arrays. First, studies of chromatin modifications provide the first ever look at regulation of gene expression within a particular brain region of a behaving animal. Second, this work has the potential of revealing fundamentally new information about the molecular mechanisms by which drugs of abuse induce particularly long-lasting changes in brain reward regions. We are using a unique strategy to overlay findings from DNA expression arrays on those from ChIP on chip arrays. The result has been a much more complete and reliable view of changes in gene expression induced in the VTA-NAc by chronic drug exposure. We are also analyzing arrays for miRNA's (microRNA's), endogenous inhibitors of mRNA expression. Drug regulation of miRNA's may explain some of the changes in steady-state mRNA levels which do not appear to be mediated via transcriptional mechanisms. The Project has recruited two biostatistic faculty who are experts in the analysis of gene array data to apply the most rigorous analysis to our large volume of array gene sets. A final effort of the Project is to investigate drug regulation of chromatin remodeling mechanisms themselves for their involvement in the process of drug addiction. Together, the advanced technologies used by this new Project offer an unprecedented evaluation of the regulation of gene expression in brain reward regions by opiates and cocaine and will guide work in the other Projects of this PPG to explore molecular and cellular mechanisms of addiction.